The Interplay between Neurotransmitters and Calcium Dynamics in Retinal Synapses during Development, Health, and Disease.

The intricate functionality of the vertebrate retina relies on the interplay between neurotransmitter activity and calcium (Ca2+) dynamics, offering important insights into developmental processes, physiological functioning, and disease progression. Neurotransmitters orchestrate cellular processes to shape the behavior of the retina under diverse circumstances. Despite research to elucidate the roles of individual neurotransmitters in the visual system, there remains a gap in our understanding of the holistic integration of their interplay with Ca2+ dynamics in the broader context of neuronal development, health, and disease. To address this gap, the present review explores the mechanisms used by the neurotransmitters glutamate, gamma-aminobutyric acid (GABA), glycine, dopamine, and acetylcholine (ACh) and their interplay with Ca2+ dynamics. This conceptual outline is intended to inform and guide future research, underpinning novel therapeutic avenues for retinal-associated disorders.

Cholesterol and PIP2 Modulation of BKCa Channels.

Ca2+/voltage-gated, large conductance K+ channels (BKCa) are formed by homotetrameric association of α (slo1) subunits. Their activity, however, is suited to tissue-specific physiology largely due to their association with regulatory subunits (ß and γ types), chaperone proteins, localized signaling, and the channel's lipid microenvironment. PIP2 and cholesterol can modulate BKCa activity independently of downstream signaling, yet activating Ca2+i levels and regulatory subunits control ligand action. At physiological Ca2+i and voltages, cholesterol and PIP2 reduce and increase slo1 channel activity, respectively. Moreover, slo1 proteins provide sites that seem to recognize cholesterol and PIP2: seven CRAC motifs in the slo1 cytosolic tail and a string of positively charged residues (Arg329, Lys330, Lys331) immediately after S6, respectively. A model that could explain the modulation of BKCa activity by cholesterol and/or PIP2 is hypothesized. The roles of additional sites, whether in slo1 or BKCa regulatory subunits, for PIP2 and/or cholesterol to modulate BKCa function are also discussed.

Embryonic Hyperglycemia Delays the Development of Retinal Synapses in a Zebrafish Model.

Embryonic hyperglycemia negatively impacts retinal development, leading to abnormal visual behavior, altered timing of retinal progenitor differentiation, decreased numbers of retinal ganglion cells and Müller glia, and vascular leakage. Because synaptic disorganization is a prominent feature of many neurological diseases, the goal of the current work was to study the potential impact of hyperglycemia on retinal ribbon synapses during embryonic development. Our approach utilized reverse transcription quantitative PCR (RT-qPCR) and immunofluorescence labeling to compare the transcription of synaptic proteins and their localization in hyperglycemic zebrafish embryos, respectively. Our data revealed that the maturity of synaptic ribbons was compromised in hyperglycemic zebrafish larvae, where altered ribeye expression coincided with the delay in establishing retinal ribbon synapses and an increase in the immature synaptic ribbons. Our results suggested that embryonic hyperglycemia disrupts retinal synapses by altering the development of the synaptic ribbon, which can lead to visual defects. Future studies using zebrafish models of hyperglycemia will allow us to study the underlying mechanisms of retinal synapse development.

Visualizing synaptic vesicle turnover and pool refilling driven by calcium nanodomains at presynaptic active zones of ribbon synapses.

Ribbon synapses of photoreceptor cells and second-order bipolar neurons in the retina are specialized to transmit graded signals that encode light intensity. Neurotransmitter release at ribbon synapses exhibits two kinetically distinct components, which serve different sensory functions. The faster component is depleted within milliseconds and generates transient postsynaptic responses that emphasize changes in light intensity. Despite the importance of this fast release for processing temporal and spatial contrast in visual signals, the physiological basis for this component is not precisely known. By imaging synaptic vesicle turnover and Ca(2+) signals at single ribbons in zebrafish bipolar neurons, we determined the locus of fast release, the speed and site of Ca(2+) influx driving rapid release, and the location where new vesicles are recruited to replenish the fast pool after it is depleted. At ribbons, Ca(2+) near the membrane rose rapidly during depolarization to levels >10 µM, whereas Ca(2+) at nonribbon locations rose more slowly to the lower level observed globally, consistent with selective positioning of Ca(2+) channels near ribbons. The local Ca(2+) domain drove rapid exocytosis of ribbon-associated synaptic vesicles nearest the plasma membrane, accounting for the fast component of neurotransmitter release. However, new vesicles replacing those lost arrived selectively at the opposite pole of the ribbon, distal to the membrane. Overall, the results suggest a model for fast release in which nanodomain Ca(2+) triggers exocytosis of docked vesicles, which are then replaced by more distant ribbon-attached vesicles, creating opportunities for new vesicles to associate with the ribbon at membrane-distal sites.

Functional roles of complexin in neurotransmitter release at ribbon synapses of mouse retinal bipolar neurons.

Ribbon synapses of photoreceptor cells and bipolar neurons in the retina signal graded changes in light intensity via sustained release of neurotransmitter. One molecular specialization of retinal ribbon synapses is the expression of complexin protein subtypes Cplx3 and Cplx4, whereas conventional synapses express Cplx1 and Cplx2. Because complexins bind to the molecular machinery for synaptic vesicle fusion (the SNARE complex) and modulate transmitter release at conventional synapses, we examined the roles of ribbon-specific complexin in regulating release at ribbon synapses of ON bipolar neurons from mouse retina. To interfere acutely with the interaction of native complexins with the SNARE complex, a peptide consisting of the highly conserved SNARE-binding domain of Cplx3 was introduced via a whole-cell patch pipette placed directly on the synaptic terminal, and vesicle fusion was monitored using capacitance measurements and FM-dye destaining. The inhibitory peptide, but not control peptides, increased spontaneous synaptic vesicle fusion, partially depleted reserve synaptic vesicles, and reduced fusion triggered by opening voltage-gated calcium channels under voltage clamp, without affecting the number of synaptic vesicles associated with ribbons, as revealed by electron microscopy of recorded terminals. The results are consistent with a dual role for ribbon-specific complexin, acting as a brake on the SNARE complex to prevent spontaneous fusion in the absence of calcium influx, while at the same time facilitating release evoked by depolarization.

Insulin treatment restores glutamate (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor function in the hippocampus of diabetic rats.

Type 1 diabetes is associated with cognitive dysfunction. Cognitive processing, particularly memory acquisition, depends on the regulated enhancement of expression and function of glutamate receptor subtypes in the hippocampus. Impairment of memory was been detected in rodent models of type 1 diabetes induced by streptozotocin (STZ). This study examines the functional properties of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and the expression of synaptic molecules that regulate glutamatergic synaptic transmission in the hippocampus of STZ-diabetic rats. The AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) and single-channel properties of synaptosomal AMPA receptors were examined after 4 weeks of diabetes induction. Results show that amplitude and frequency of mEPSCs recorded from CA1 pyramidal neurons were decreased in diabetic rats. In addition, the single-channel properties of synaptic AMPA receptors from diabetic rat hippocampi were different from those of controls. These impairments in synaptic currents gated by AMPA receptors were accompanied by decreased protein levels of AMPA receptor subunit GluR1, the presynaptic protein synaptophysin, and the postsynaptic anchor protein postsynaptic density protein 95 in the hippocampus of diabetic rats. Neural cell adhesion molecule (NCAM), an extracellular matrix molecule abundantly expressed in the brain, and the polysialic acid (PSA) attached to NCAM were also downregulated in the hippocampus of diabetic rats. Insulin treatment, when initiated at the onset of diabetes induction, reduced these effects. These findings suggest that STZ-induced diabetes may result in functional deteriorations in glutamatergic synapses in the hippocampus of rats and that these effects may be reduced by insulin treatment.

Stabilization of spontaneous neurotransmitter release at ribbon synapses by ribbon-specific subtypes of complexin.

Ribbon synapses of tonically releasing sensory neurons must provide a large pool of releasable vesicles for sustained release, while minimizing spontaneous release in the absence of stimulation. Complexins are presynaptic proteins that may accomplish this dual task at conventional synapses by interacting with the molecular machinery of synaptic vesicle fusion at the active zone to retard spontaneous vesicle exocytosis yet facilitate release evoked by depolarization. However, ribbon synapses of photoreceptor cells and bipolar neurons in the retina express distinct complexin subtypes, perhaps reflecting the special requirements of these synapses for tonic release. To investigate the role of ribbon-specific complexins in transmitter release, we combined presynaptic voltage clamp, fluorescence imaging, electron microscopy, and behavioral assays of photoreceptive function in zebrafish. Acute interference with complexin function using a peptide derived from the SNARE-binding domain increased spontaneous synaptic vesicle fusion at ribbon synapses of retinal bipolar neurons without affecting release triggered by depolarization. Knockdown of complexin by injection of an antisense morpholino into zebrafish embryos prevented photoreceptor-driven migration of pigment in skin melanophores and caused the pigment distribution to remain in the dark-adapted state even when embryos were exposed to light. This suggests that loss of complexin function elevated spontaneous release in illuminated photoreceptors sufficiently to mimic the higher release rate normally associated with darkness, thus interfering with visual signaling. We conclude that visual system-specific complexins are required for proper illumination-dependent modulation of the rate of neurotransmitter release at visual system ribbon synapses.

The ribbon-associated protein C-terminal-binding protein 1 is not essential for the structure and function of retinal ribbon synapses.

PURPOSE: Synaptic ribbons are organelles found at presynaptic active zones of sensory neurons that generate sustained graded electrical signals in response to stimuli, including retinal photoreceptor cells and bipolar neurons. RIBEYE is the major and specific protein constituent of ribbons; however, over the past decade an increasing number of other proteins have been identified at ribbon active zones, including C-terminal-binding protein 1 (CtBP1; a regulator of transcription and membrane trafficking that might bind to the B domain of RIBEYE). The appearance of CtBP1 together with RIBEYE suggests that it may contribute to ribbon function, but the possible role of CtBP1 at ribbon synapses has not yet been examined. Using CtBP1-knockout mice, we tested for functional effects of absence of CtBP1 protein. METHODS: Confocal microscopy, electrophysiology, and electron microscopy were used to examine the structure and function of ribbon synapses in the retina and in isolated bipolar neurons from CtBP1 null mice compared with their wild-type littermates. RESULTS: Expression of ribbons appeared to be normal in CtBP1 null mouse retina as revealed by immunofluorescence with an antibody to the B domain of RIBEYE and by binding studies using a fluorescent peptide that binds to RIBEYE in ribbons of living bipolar cells. Electron microscopy also showed grossly normal pre- and postsynaptic organization of ribbon synapses in both photoreceptors and bipolar cells. Synaptic vesicles were normal in size, but the overall density of reserve vesicles was reduced by ~20% in the cytoplasm of CtBP1 null ribbon synaptic terminals. However, the reduced vesicle density did not detectably alter synaptic function of bipolar neurons as revealed by activity-dependent loading of synaptic vesicles with FM4-64, presynaptic calcium current, capacitance measurements of synaptic exocytosis, and destaining of FM dye upon stimulation. CONCLUSIONS: Overall the results suggest that CtBP1 protein is not essential for the formation of functional ribbon synapses in the retina.

Tauroursodeoxycholic Acid Alleviates Endoplasmic Reticulum Stress-Mediated Visual Deficits in Diabetic tie2-TNF Transgenic Mice via TGR5 Signaling.

Purpose: This study evaluated if tauroursodeoxycholic acid (TUDCA) alleviates pro-inflammatory and endoplasmic reticulum (ER) stress-mediated visual deficits in diabetic tie2-TNF transgenic mice via Takeda G protein-coupled receptor 5 (TGR5) receptor signaling. Methods: Adult tie2-TNF transgenic or age-matched C57BL/6J (wildtype, WT) mice were made diabetic and treated subcutaneously with TUDCA. After 4 weeks, visual function, vascular permeability, immunohistology, and molecular analyses were assessed. Human retinal endothelial cells (HRECs) silenced for TGR5, followed by TNF and high glucose (HG) stress-mediated endothelial permeability, and transendothelial migration of activated leukocytes were assessed with TUDCA in vitro. Results: Compared with WT mice, tie2-TNF mice showed a decreased visual function correlated with a decrease in protein kinase C α (PKCα) in rod bipolar cells, and increased vascular permeability was further exacerbated in diabetic-tie2-TNF mice. Conversely, TUDCA alleviated these changes in diabetic mice. An increase in inflammation and ER stress in retina coincided with an increase in TGR5 expression in diabetic tie2-TNF mice that decreased with TUDCA. In vitro, HRECs exposed to TNF+HG demonstrated >2-fold increase in TGR5 expression, a 3-fold increase in leukocyte transmigration with a concomitant increase in permeability. Although TUDCA reversed these effects, HRECs silenced for TGR5 and challenged with TUDCA or TGR5 agonist failed to reverse the TNF+HG induced effects. Conclusions: Our data suggest that TUDCA will serve as an excellent therapeutic agent for diabetic complications addressing both vascular and neurodegenerative changes in the retina. Perturbation of the TGR5 receptor in the retina might play a role in linking retinal ER stress to neurovascular dysfunction in diabetic retinopathy.

The Effects of Aging on Rod Bipolar Cell Ribbon Synapses.

The global health concern posed by age-related visual impairment highlights the need for further research focused on the visual changes that occur during the process of aging. To date, multiple sensory alterations related to aging have been identified, including morphological and functional changes in inner hair cochlear cells, photoreceptors, and retinal ganglion cells. While some age-related morphological changes are known to occur in rod bipolar cells in the retina, their effects on these cells and on their connection to other cells via ribbon synapses remain elusive. To investigate the effects of aging on rod bipolar cells and their ribbon synapses, we compared synaptic calcium currents, calcium dynamics, and exocytosis in zebrafish (Danio rerio) that were middle-aged (MA,18 months) or old-aged (OA, 36 months). The bipolar cell terminal in OA zebrafish exhibited a two-fold reduction in number of synaptic ribbons, an increased ribbon length, and a decrease in local Ca2+ signals at the tested ribbon location, with little change in the overall magnitude of the calcium current or exocytosis in response to brief pulses. Staining of the synaptic ribbons with antibodies specific for PKCa revealed shortening of the inner nuclear and plexiform layers (INL and IPL). These findings shed light on age-related changes in the retina that are related to synaptic ribbons and calcium signals.

The role of sphingosine-1-phosphate receptor 2 in mouse retina light responses.

The bioactive sphingolipid sphingosine-1-phosphate (S1P) acts as a ligand for a family of G protein-coupled S1P receptors (S1PR1-5) to participate in a variety of signaling pathways. However, their specific roles in the neural retina remain unclear. We previously showed that S1P receptor subtype 2 (S1PR2) is expressed in murine retinas, primarily in photoreceptors and bipolar cells, and its expression is altered by retinal stress. This study aims to elucidate the role of S1PR2 in the mouse retina. We examined light responses by electroretinography (ERG), structural differences by optical coherence tomography (OCT), and protein levels by immunohistochemistry (IHC) in wild-type (WT) and S1PR2 knockout (KO) mice at various ages between 3 and 6 months. We found that a- and b-wave responses significantly increased at flash intensities between 400∼2000 and 4∼2,000 cd.s/m 2 respectively, in S1PR2 KO mice relative to those of WT controls at baseline. S1PR2 KO mice also exhibited significantly increased retinal nerve fiber layer (RNFL) and outer plexiform layer (OPL) thickness by OCT relative to the WT. Finally, in S1PR2 KO mice, we observed differential labeling of synaptic markers by immunohistochemistry (IHC) and quantitative reverse transcription polymerase chain reaction (RT-qPCR). These results suggest a specific involvement of S1PR2 in the structure and synaptic organization of the retina and a potential role in light-mediated functioning of the retina.

The Role of Sphingosine-1-Phosphate Receptor 2 in Mouse Retina Light Responses.

The bioactive sphingolipid sphingosine-1-phosphate (S1P) acts as a ligand for a family of G protein-coupled S1P receptors (S1PR1-5) to participate in a variety of signaling pathways. However, their specific roles in the neural retina remain unclear. We previously showed that S1P receptor subtype 2 (S1PR2) is expressed in murine retinas, primarily in photoreceptors and bipolar cells, and its expression is altered by retinal stress. This study aims to elucidate the role of S1PR2 in the mouse retina. We examined light responses by electroretinography (ERG), structural differences by optical coherence tomography (OCT), and protein levels by immunohistochemistry (IHC) in wild-type (WT) and S1PR2 knockout (KO) mice at various ages between 3 and 6 months. We found that a- and b-wave responses significantly increased at flash intensities between 400~2000 and 4~2000 cd.s/m2, respectively, in S1PR2 KO mice relative to those of WT controls at baseline. S1PR2 KO mice also exhibited significantly increased retinal nerve fiber layer (RNFL) and outer plexiform layer (OPL) thickness by OCT relative to the WT. Finally, in S1PR2 KO mice, we observed differential labeling of synaptic markers by immunohistochemistry (IHC) and quantitative reverse transcription polymerase chain reaction (RT-qPCR). These results suggest a specific involvement of S1PR2 in the structure and synaptic organization of the retina and a potential role in light-mediated functioning of the retina.

Smooth muscle cholesterol enables BK ß1 subunit-mediated channel inhibition and subsequent vasoconstriction evoked by alcohol.

OBJECTIVE: Hypercholesterolemia and alcohol drinking constitute independent risk factors for cerebrovascular disease. Alcohol constricts cerebral arteries in several species, including humans. This action results from inhibition of voltage- and calcium-gated potassium channels (BK) in vascular smooth muscle cells (VSMC). BK activity is also modulated by membrane cholesterol. We investigated whether VSMC cholesterol regulates ethanol actions on BK and cerebral arteries. METHODS AND RESULTS: After myogenic tone development, cholesterol depletion of rat, resistance-size cerebral arteries ablated ethanol-induced constriction, a result that was identical in intact and endothelium-free vessels. Cholesterol depletion reduced ethanol inhibition of BK whether the channel was studied in VSMC or after rat cerebral artery myocyte subunit (cbv1+ß1) reconstitution into phospholipid bilayers. Homomeric cbv1 channels reconstituted into bilayers and VSMC BK from ß1 knockout mice were both resistant to ethanol-induced inhibition. Moreover, arteries from ß1 knockout mice failed to respond to ethanol even when VSMC cholesterol was kept unmodified. Remarkably, ethanol inhibition of cbv1+ß1 in bilayers and wt mouse VSMC BK were drastically blunted by cholesterol depletion. Consistently, cholesterol depletion suppressed ethanol constriction of wt mouse arteries. CONCLUSION: VSMC cholesterol and BK ß1 are both required for ethanol inhibition of BK and the resulting cerebral artery constriction, with health-related implications for manipulating cholesterol levels in alcohol-induced cerebrovascular disease.

Tracking the dynamics of single fused synaptic vesicle proteins from a single ribbon active zone in zebrafish retinal bipolar cells.

Clearance of fused synaptic vesicle components and availability of release sites are important determinants of recovery from short-term synaptic depression. However, the dynamics of release site clearance are not well established. This protocol illustrates single-molecule imaging of an exocytosis reporter, synaptophysin-pHluorin fusion protein (SypHy), by combining two-color laser scanning confocal microscopy with whole-cell patch-clamp recording of retinal bipolar cells from transgenic zebrafish that weakly express SypHy to track the dynamics of newly fused vesicle proteins at the active zone. For complete details on the use and execution of this profile, please refer to Vaithianathan et al. (2019).

Subtype identification and functional characterization of ryanodine receptors in rat cerebral artery myocytes.

Ryanodine receptors (RyRs) regulate contractility in resistance-size cerebral artery smooth muscle, yet their molecular identity, subcellular location, and phenotype in this tissue remain unknown. Following rat resistance-size cerebral artery myocyte sarcoplasmic reticulum (SR) purification and incorporation into POPE-POPS-POPC (5:3:2; wt/wt) bilayers, unitary conductances of 110 +/- 8, 334 +/- 15, and 441 +/- 27 pS in symmetric 300 mM Cs(+) were usually detected. The most frequent (34/40 bilayers) conductance (334 pS) decreased to

Channel beta2-4 subunits fail to substitute for beta1 in sensitizing BK channels to lithocholate.

Large conductance, calcium- and voltage-gated potassium (BK) channels regulate numerous physiological processes. While most basic functional characteristics of native BK channels are reproduced by BK alpha (slo1) subunit homotetramers, key biophysical and pharmacological properties are drastically modified by the presence of auxiliary beta subunits (encoded by KCNMB1-4). Numerous physiological steroids, including sex hormones, gluco- and mineralocorticoids, activate beta subunit-containing BK channels, yet these steroids appear to be sensed by different types of beta subunits, with some steroids being sensed by homomeric slo1 channels as well. We recently showed that beta1 sensitizes the BK channel to microM concentrations of lithocholate (LC). Following expression of rat cerebral artery myocyte slo1 subunits ("cbv1") with beta1, beta2, beta3 or beta4 in Xenopus laevis oocytes we now demonstrate that BK beta2, beta3 and beta4 subunits fail to substitute for beta1 in providing LC-sensitivity (150 microM) to the BK channel. These findings document for the first time a rather selective steroid activation of BK channels via a particular channel accessory subunit. In addition, LC routinely activated native BK channels in myocytes freshly isolated from rat cerebral artery smooth muscle, where BK beta1 is highly expressed, while failing to do so in skeletal (flexor digitorum brevis) muscle, where BK beta1 expression is negligible. This indicates that the native environment of the BK channel sustains the LC-sensitivity distinctly provided to the BK channel by beta1 subunits. Our study indicates that LC represents a unique tool to probe the presence of functional beta1-subunits and selectively activate BK channels in tissues that highly express KCNMB1.

Tracking Newly Released Synaptic Vesicle Proteins at Ribbon Active Zones.

Clearance of synaptic vesicle proteins from active zones may be rate limiting for sustained neurotransmission. Issues of clearance are critical at ribbon synapses, which continually release neurotransmitters for prolonged periods of time. We used synaptophysin-pHluorin (SypHy) to visualize protein clearance from active zones in retinal bipolar cell ribbon synapses. Depolarizing voltage steps gave rise to small step-like changes in fluorescence likely indicating release of single SypHy molecules from fused synaptic vesicles near active zones. Temporal and spatial fluorescence profiles of individual responses were highly variable, but ensemble averages were well fit by clearance via free diffusion using Monte Carlo simulations. The rate of fluorescence decay of ensemble averages varied with the time and location of the fusion event, with clearance being most rapid at the onset of a stimulus when release rate is the highest.

Ethanol modulates BKCa channels by acting as an adjuvant of calcium.

Ethanol modulation of calcium- and voltage-gated potassium (slo1) channels alters neuronal excitability, cerebrovascular tone, brain function, and behavior, yet the mechanism of this modulation remains unknown. Using patch-clamp electrophysiology on recombinant BK(Ca) channels cloned from mouse brain and expressed in Xenopus laevis oocytes, we demonstrate that ethanol, even at concentrations maximally effective to modulate BK(Ca) channel function (100 mM), fails to gate the channel in absence of activating calcium. Moreover, ethanol does not modify intrinsic, voltage- or physiological magnesium-driven gating. The alcohol works as an adjuvant of calcium by selectively facilitating calcium-driven gating. This facilitation, however, renders differential ethanol effects on channel activity: potentiation at low (<10 microM) and inhibition at high (>10 microM) calcium, this dual pattern remaining largely unmodified by coexpression of brain slo1 channels with the neuronally abundant BK(Ca) channel beta(4) subunit. Calcium recognition by either of the slo1 high-affinity sensors (calcium bowl and RCK1 Asp362/Asp367) is required for ethanol to amplify channel activation by calcium. The Asp362/Asp367 site, however, is necessary and sufficient to sustain ethanol inhibition. This inhibition also results from ethanol facilitation of calcium action; in this case, ethanol favors channel dwelling in a calcium-driven, low-activity mode. The agonist-adjuvant mechanism that we advance from the calcium-ethanol interaction on slo1 might be applicable to data of ethanol action on a wide variety of ligand-gated channels.

The second transmembrane domain of the large conductance, voltage- and calcium-gated potassium channel beta(1) subunit is a lithocholate sensor.

Bile acids and other steroids modify large conductance, calcium- and voltage-gated potassium (BK) channel activity contributing to non-genomic modulation of myogenic tone. Accessory BK beta(1) subunits are necessary for lithocholate (LC) to activate BK channels and vasodilate. The protein regions that sense steroid action, however, remain unknown. Using recombinant channels in 1-palmitoyl-2-oleoyl-phosphatidylethanolamine/1-palmitoyl-2-oleoyl-phosphatidylserine bilayers we now demonstrate that complex proteolipid domains and cytoarchitecture are unnecessary for beta(1) to mediate LC action; beta(1) and a simple phospholipid microenvironment suffice. Since beta(1) senses LC but beta(4) does not, we made chimeras swapping regions between these subunits and, following channel heterologous expression, demonstrate that beta(1) TM2 is a bile acid-recognizing sensor.

Lysosomal dysfunction produces distinct alterations in synaptic alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid and N-methyl-D-aspartate receptor currents in hippocampus.

The early processes that lead to synaptic dysfunction during aging are not clearly understood. Dysregulation of alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors may cause age-related cognitive decline. Using hippocampal slice cultures exhibiting lysosomal dysfunction, an early marker of brain aging that is linked to protein accumulation, we identified alterations to AMPA and NMDA receptor-mediated synaptic currents. The miniature and spontaneous excitatory postsynaptic currents that were examined after 3, 6, and 9 days of lysosomal disruption showed progressive changes in amplitude, frequency, and rise and decay kinetics. To investigate whether modifications in specific channel properties of single synaptic receptors contributed to changes in the amplitude and time course of synaptic currents, we examined the single channel properties of synaptic AMPA and NMDA receptors. The channel open probability and the mean open times showed decreases in both receptor populations, whereas the closed times were increased without any change in the channel conductance. The Western blot analysis revealed a progressive decline in synaptic markers including glutamate receptor subunits. These results indicate that lysosomal dysfunction leads to progressive functional perturbation of AMPA and NMDA receptors in this slice model of protein accumulation, suggesting that age-related cognitive decline could result from altered glutamate receptor function before reductions in synaptic density.